BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 10 ppsx

synthesize chemically and display a biological half-life of up to 24 h. Most antisense oligos currently being assessed in clinical trials are S-oligos. Delivery and cellular uptake of oligonucleotides Oligo administration during many clinical trials entails direct i.v. infusion, often over a course of several hours. Although relatively stable in serum, the commonly employed phospho rothioate oligos (and indeed most other oligo types) encounter several barriers to reaching their final destinations. They bind various serum proteins, including serum albumin, as well as a range of heparin-binding and other proteins, which commonly occur on many cell surfaces. Targeting of naked oligos to specific cell types is therefore not possible. Following administration, these oligos tend to be distributed to many tissues, with the highest pr oportion accumulating in the liver and kidney. The precise mechanism(s) by which oligos enter cells are not fully understood. Most are charged molecules, sometimes displaying a molecular mass of up to 10–12 kDa. Receptor- mediated endocytosis appears to be the most common mechanism by which charged oligos, such as phosphorothi oates, enter most cells. One putative phosphorothioate receptor appears to consist of an 80 kDa surface protein, associated with a smaller 34 kDa membrane protein. However, this in itself seems to be an inefficient process, with only a small proportion of the administered drug eventually being transferred across the plasma membrane. Uncharged oligos appear to enter the cell by passive diffusion, as well as possibly by endocytosis. However, elimination of the charges renders the resultant oligos relatively hydrophobic, thus generating additional difficulties with their synthesis and delivery. Attempts to increase delivery of oligos into the cell centre mainly on the use of suitable carrier systems. Liposomes, as well as polymeric carriers (e.g. polylysine-based carriers), are gaining most attention in this regard. Details of such carriers have already been discussed earlier in this chapter. An alternative system, which effectively results in the introduction of antisense oligonucleo- tides into the cell, entails the application of gene therapy. In this case, a gene which, when transcribed, yields (antisense) mRNA of appropriate nucleotide sequence, is introduced into the cell by a retroviral or other appropriate vector. This approach, as applied to the treatment of cancer and AIDS, is being appraised in a number of trials. Oligos, including modified oligos, appear to be ultimately metabolized within the cell by the action of nucleases, particularly 3’-exonucleases. Breakdown metabolic produ cts are then mainly excreted via the urinary route. Manufacture of oligonucleotides In contrast to the biopharmaceuticals thus far discussed (recombinant proteins and gene therapy plasmids), antisense oligonucleotides are manufactured by direct chemical synthesis. Organic synthetic pathways have been developed, optimized and commercialized for some time, as oligonucleotides are widely used reagents in molecular biology. They are required as primers, probes and for the purposes of site-directed mutagenesis. The basic synthetic strategy is very similar in concept to the means by which peptides are synthesized via the Merrifield method, as described in Chapter 2 (Box 2.1). The nucleotides required (themselves either modified or unmodified, as desired) are first reacted with a protecting chemical group. Each protected nucleotide is then coupled in turn to the growing end of the nucleotide chain, itself attached to a NUCLEIC ACID THERAPEUTICS 493 solid phase. After coupling, the original protecting group is removed and, when chain synthesis is complete, the bond anchoring the chemical to the solid phase is hydrolysed, releasing the free oligo. This may then be purified by HPLC. The most common synthetic method used is known as the phosphoramidite method, which uses a dimethox ytrityl (DMTr) protecting group and tetrazole as the coupling agent. Automated synthesizers are commercially available which can quickly and inexpensively synthesize oligos of over 100 nucleotides. Vitravene, an approved antisense agent On 26 August 1998, Vitravene became the first (and thus far apparently the only) antisense product to be approved for general medical use by the FDA. It gained approval within the European Union the following year, although it has since been withdrawn from the EU market for commer cial rather than technical reasons. Vitravene is the trade name given to a 21- nucleotide phosphorothioate based product of the following base sequence: 5’-G–C–G–T–T–T–G–C–T–C–T–T–C–T–T–C–T–T–G–C–G-3’ Developed by the US company Isis, Vitravene is used to treat cytomegalovirus (CMV) retinitis in AIDS patients. It is formulated as a sterile solution in WFI (Chapter 3) using a bicarbonate buffer to maintain a final product pH of 8.7. Administration is by direct injection into the eye (intravitreal injection) and each ml of product contains 6.6 mg of active ingredient. The product inhibi ts replication of human cytomegalovirus (HCMV) via an antisense mechanism. Its nucleotide sequence is complementary to a sequence in mRNA transcripts of the major immediate early region (IE2 region) of HCMV. These mRNAs cod e for several essential viral proteins and blocking their synthesis effectively inhibits viral replication. Antigene sequences and ribozymes Antigene sequences and ribozymes form two additional classes of antisense agents. However, the therapeutic potential of these agents is only now beginning to be appraised. Certain RNA sequences can function as catalysts. These so-called ‘ribozymes’ function to catalyse cleavage at specific sequences in a specific mRNA substrate. Many ribozymes will cleave their target mRNA where there exists a particular triplet nucleotide sequence G–U–C. Statistically, it is likely that this triplet will occur at least once in most mRNAs. Ribozymes can be directed to a specific mRNA by introducing short-flanking oligonucleo- tides, which are complementary to the target mRNA (Figure 11.15). The resultant cleavage of the target obviously preven ts translation. One potential advantage of ribozymes is that, as catalytic agents, a single molecule could likely destroy thousands of copies of the target mRNA. Such a drug should, therefore, be very potent. ‘Antigene’ (oligonucleotide) sequences function to inhibit transcription of a specific gene (as opposed to inhibition of translation of a mRNA species). These oligonucleotides achieve this by hybridizing with appropriate stretches of double-stranded DNA, forming a triple helix. This inhibits initiation of transcription of the genes in this region. The binding of antigene sequences occurs only in the so-called ‘major groove’ of DNA. The incoming oligonucleotide does not disrupt the double-stranded DNA. It binds to it, forming what are termed ‘Hoogsteen base pairs’ — each base in the antigene sequence forming two new 494 BIOPHARMACEUTICALS hydrogen bonds with a purine base in the targeted region of the double helix. Much research, however, must be undertaken before it will become clear whether such antigene sequences will be of therapeutic use. CONCLUSION Every few decades, a medical innovation is perfected that profoundly influences the practice of medicine. Widespread vaccina tion against common infectious agents and the discovery of antibiotics serve as two such examples. Many scientists now believe that the potential of gene therapy and antisense technology rivals even the most significant medical advances achieved to date. It is now just over a decade since the first nucleic acid-based drugs began initial tests. Several such drugs will likely be in routine medical use in less than one decade more. The application of gene technol ogy could also change utterly the profile of biopharmaceutical drugs currently on the market. Virtually all such products are proteins, currently administered to patients for short or prolonged periods, as appropriate. Gene therapy offers the possibility of equipping the patient’s own body with the ability to synthesize these drugs itself, and over whatever time scale is appropriate. Taken to its logical conclusion, gene therapy thus offers the potential to render obsolete most of the biopharmaceutical products currently on the market. Of all the biopharmaceuticals discussed throughout this text, nucleic acid-based drugs may well turn out to have the most profound influence on the future practice of molecular medicine. NUCLEIC ACID THERAPEUTICS 495 Figure 11.15. Outline of how ribozyme technology could prevent translation of specific mRNA, thus preventing synthesis of a specific target protein Flanking sequences which ‘dock’ ribozyme at the appropriate sequence of the appropriate mRNA via complementary base pairing Flanking sequences which ‘dock’ ribozyme at the appropriate sequence of the appropriate mRNA via complementary base pairing FURTHER READING Books Blankenstein, T. (Ed.) (1999). Gene Therapy: Principles and Applications. Birkhauser-Verlag. Crooke, S. (Ed.) (2001). Antisense Drug Technology. Marcel Dekker, New York. Kresina, T. (Ed.) (2001). An Introduction to Molecular Medicine and Gene Therapy, Parts I and II. Wiley-Liss, New York. Lowrie, D. (1999). DNA Vaccines. Humana, New York. Phillips, M. (2000). Antisense Technology (Methods in Enzymology, Vol. 313). Academic Press, New York. Stein, C. & Krieg, A. (1998). Applied Antisense Oligonucleotide Technology. Wiley, Chichester. Articles Gene therapy Buchschacher, G. & Wong-Staal, F. (2001). Approaches to gene therapy for human immunodeficiency virus infection. Human Gene Ther. 12(9), 1013–1019. Davies, J. et al. (2001). Gene therapy for cystic fibrosis. J. Gene Med. 3(5), 409–417. Demeterco, C. & Levine, F. (2001). Gene therapy for diabetes. Frontiers Biosci. 6, D175–D191. Demoly, P. et al. (1997). Gene therapy strategies for asthma. Gene Therapy 4(6), 507–516. Docherty, K. (1997). Gene therapy for diabetes mellitus. Clin. Sci. 92(4), 321–330. Donnelly, J. (1997). DNA vaccines. Ann. Rev. Immunol. 15, 617–648. Felgner, P. (1997). Nonviral strategies for gene therapy. Sci. Am. June, 86–90. Ferreira, G. et al. (2000). Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. Trends Biotechnol. 18(9), 380–388. Lewin, A. & Hauswirth, W. (2001). Ribozyme gene therapy: applications for molecular medicine. Trends Mol. Med. 7(5), 221–228. Liras, A. (2001). Gene therapy for haemophilia: the end of a ‘royal pathology’ in the third millennium? Haemophilia 7(5), 441–445. Mhashilkar, A. et al. (2001). Gene therapy — therapeutic approaches and implications. Biotechnol. Adv. 19(4), 279–297. Moller, P. & Schadendorf, D. (1997). Somatic gene therapy and its implications in melanoma treatment. Arch. Dermatol. Res. 289(2), 71–77. Mulligan, R. (1993). The basic science of gene therapy. Science 260, 926–931. Pfeifer, A. & Verma, I. (2001). Gene therapy: promises and problems. Ann. Rev. Genom. Hum. Genet. 2, 177–211. Phillips, A. (2001). The challenge of gene therapy and DNA delivery. J. Pharm. Pharmacol. 53(9), 1169–1174. Robertson, J. & Griffiths, E. (2001). Assuring the quality, safety and efficacy of DNA vaccines. Mol. Biotechnol. 17(2), 143–149. Rosenberg, S. (1997). Cancer vaccines based on the identification of genes encoding cancer regression antigens. Immunol. Today 18(4), 175–182. Schatzlein, A. (2001). Non-viral vectors in cancer gene therapy: principles and progress. Anti-cancer Drugs 12(4), 275–304. Scott-Taylor, T. & Dalgeish, A. (2000). DNA vaccines. Expert Opin. Invest. Drugs 9(3), 471–480. Smith, A. (1995). Viral vectors in gene therapy. Ann. Rev. Microbiol. 49, 807–838. Smith, H. & Klinman, D. (2001). The regulation of DNA vaccines. Curr. Opin. Biotechnol. 12(3), 299–303. Wu, N. & Ataai, M. (2000). Production of viral vectors for gene therapy applications. Curr. Opin. Biotechnol. 11(2), 205–208. Antisense technology Adah, S. et al. (2001). Chemistry and biochemistry of 2’-5’ oligoadenylate-based antisense strategy. Cur. Med. Chem. 8(10), 1189–1212. Akhtar, S. et al. (2000). The delivery of antisense therapeutics. Adv. Drug Delivery Rev. 44(1), 3–21. Askari, F. (1996). Molecular medicine: antisense-oligonucleotide therapy. N. Engl. J. Med. 334(5), 316–318. Galderisi, U. et al. (2001). Antisense oligonucleotides as drugs for HIV treatment. Expert Opin. Therapeut. Patents 11(10), 1605–1611. Hughes, M. et al. (2001). The cellular delivery of antisense oligonucleotides and ribozymes. Drug Discovery Today 6(6), 303–315. Lebedeva, I. & Stein, C. (2001). Antisense oligonucleotides: promise and reality. Ann. Rev. Pharmacol. Toxicol. 41, 403–419. Pawlak, W. et al. (2000). Antisense therapy in cancer. Cancer Treatment Rev. 26(5), 333–350. 496 BIOPHARMACEUTICALS Reddy, D. (1996). Antisense oligonucleotides: a new class of potential anti-AIDS and anti-cancer drugs. Drugs Today 32(2), 113–137. Taylor, M. (2001). Emerging antisense technologies for gene functionalization and drug discovery. Drug Discovery Today 6(15), S97–S101. Wagner, R. & Flanagan, W. (1997). Antisense technology and prospects for therapy of viral infections and cancer. Mol. Med. Today 3(1), 31–38. Wickstrom, E. (1992). Strategies for administering targeted therapeutic oligodeoxynucleotides. Trends Biotechnol. 10, 281–286. NUCLEIC ACID THERAPEUTICS 497 Appendix 1 Biopharmaceuticals thus far approved in the USA or European Union Notes: (a) Several products have been approved for multiple indications. Only the first indication for which each was approved is listed. (b) ‘Vet’ listing in therapeutic indication column indicates an animal application. All other products are used in human medicine. Abbreviations: r¼recombinant, rh¼recombi nant human, CHO¼Chinese hamster ovary, BHK¼baby hamster kidney, Mab¼monoclonal antibody, tPA¼ tissue plasminogen activator, hGH¼human growth hormone, FSH ¼follicle stimulating hormone, TSH ¼thyroid stimulating hormone, EPO¼erythropoietin, GM-CSF¼granulocyte-macrophage colony stimulating factor, IFN¼interferon, IL ¼interleukin, HBsAg¼ hepatitis B surface antigen, PDGF¼ platelet-derived growth factor, TNFR ¼ tumour necrosis factor receptor, E. coli ¼Escherichia coli, S. cerevisiae¼Saccharomyces cerevisiae. Product Company Therapeutic indication Date approved Recombinant blood factors Bioclate (rhFactor VIII produced in CHO cells) Centeon Haemophilia A 1993 (USA) Benefix (rhFactor IX produced in CHO cells) Genetics Institute Haemophilia B 1997 (USA, EU) Kogenate (rhFactor VIII produced in BHK cells. Also sold as Helixate by Centeon via a license agreement) Bayer Haemophilia A 1993 (USA), 2000 (EU) Helixate NexGen (octocog-a; rhFactor VIII produced in BHK cells) Bayer Haemophilia A 2000 (EU) NovoSeven (rhFactor VIIa produced in BHK cells) Novo Nordisk Some forms of haemophilia 1995 (EU), 1999 (USA) (Continued) Biopharmaceuticals: Biochemistry and Biotechnology, Second Edition by Gary Walsh John Wiley & Sons Ltd: ISBN 0 470 84326 8 (ppc), ISBN 0 470 84327 6 (pbk) Product Company Therapeutic-indication Date approved Recombinate (rhFactor VIII produced in an animal cell line) Baxter Healthcare/ Genetics Institute Haemophilia A 1992 (USA) ReFacto (Moroctocog-a, i.e. B-domain-deleted rhFactor VIII produced in CHO cells) Genetics Institute Haemophilia A 1999 (EU), 2000 (USA) Recombinant tissue plasminogen activator-based products Activase (Alteplase, rh-tPA produced in CHO cells) Genentech Acute myocardial infarction 1987 (USA) Ecokinase (Reteplase, rtPA; differs from human tPA in that three of its five domains have been deleted. Produced in E. coli ) Galenus Mannheim Acute myocardial infarction 1996 (EU) Retavase (Reteplase, rtPA; see Ecokinase) Boehringer- Mannheim/ Centocor Acute myocardial infarction 1996 (USA) Rapilysin (Reteplase, rtPA; see Ecokinase) Boehringer- Mannheim Acute myocardial infarction 1996 (EU) Tenecteplase (also marketed as Metalyse) (TNK-tPA, modified rtPA produced in CHO cells) Boehringer- Ingelheim Myocardial infarction 2001 (EU) TNKase (Tenecteplase; modified rtPA produced in CHO cells; see Tenecteplase) Genentech Myocardial infarction 2000 (USA) Recombinant hormones Humulin (rhInsulin produced in E. coli ) Eli Lilly Diabetes mellitus 1982 (USA) Novolin (rhInsulin) Novo Nordisk Diabetes mellitus 1991 (USA) Humalog (Insulin Lispro, an insulin analogue produced in E. coli ) Eli Lilly Diabetes mellitus 1996 (USA, EU) Insuman (rhInsulin produced in E. coli ) Hoechst AG Diabetes mellitus 1997 (EU) Liprolog (Bio Lysprol, a short- acting insulin analogue produced in E. coli ) Eli Lilly Diabetes mellitus 1997 (EU) NovoRapid (Insulin Aspart, short- acting rhInsulin analogue) Novo Nordisk Diabetes mellitus 1999 (EU) Novomix 30 (contains Insulin Aspart, short acting rhInsulin analogue — see NovoRapid — as one ingredient) Novo Nordisk Diabetes mellitus 2000 (EU) Novolog (Insulin Aspart, short- acting rhInsulin analogue produced in S. cerevisiae. See also Novorapid) Novo Nordisk Diabetes mellitus 2001 (USA) Novolog mix 70/30 (contains Insulin Aspart, short-acting rhInsulin analogue, as one ingredient. See also Novomix 30) Novo Nordisk Diabetes mellitus 2001 (USA) 500 BIOPHARMACEUTICALS Product Company Therapeutic indication Date approved Actrapid/Velosulin/Monotard/ Insulatard/Protaphane/Mixtard/ Actraphane/Ultratard (All contain rhInsulin produced in S. cerevisiae, formulated as short–intermediate– long-acting products) Novo Nordisk Diabetes mellitus 2002 (EU) Lantus (Insulin glargine, long- acting rhInsulin analogue produced in E. coli) Aventis Pharmaceuticals Diabetes mellitus 2000 (USA, EU) Optisulin (Insulin glargine, long-acting rhInsulin analogue produced in E. coli. See Lantus) Aventis Pharmaceuticals Diabetes mellitus 2000 (EU) Protropin (rhGH, differs from human hormone only by containing an additional N-terminal methionine residue. Produced in E. coli) Genentech hGH deficiency in children 1985 (USA) Glucagen (rhGlucagon. produced in S. cerevisiae) Novo Nordisk Hypoglycaemia 1998 (USA) Thyrogen (Thyrotrophin-a, rhTSH produced in CHO cells) Genzyme Detection/treatment of thyroid cancer 1998 (USA), 2000 (EU) Humatrope (rhGH, produced in E. coli ) Eli Lilly hGH deficiency in children 1987 (USA) Nutropin (rhGH, produced in E. coli ) Genentech hGH deficiency in children 1994 (USA) Nutropin AQ (rhGH, produced in E. coli ) Schwartz Pharma AG Growth failure, Turner’s syndrome 2001 (EU) BioTropin (rhGH) Biotechnology General hGH deficiency in children 1995 (USA) Genotropin (rhGH, produced in E. coli ) Pharmacia and Upjohn hGH deficiency in children 1995 (USA) Saizen (rhGH) Serono Laboratories hGH deficiency in children 1996 (USA) Serostim (rhGH) Serono Laboratories Treatment of AIDS- associated catabolism/ wasting 1996 (USA) Norditropin (rhGH) Novo Nordisk Treatment of growth failure in children due to inadequate growth hormone secretion 1995 (USA) Gonal F (rhFSH, produced in CHO cells) Serono Anovulation and superovulation 1995 (EU), 1997 (USA) Puregon (rhFSH, produced in CHO cells) N.V. Organon Anovulation and superovulation 1996 (EU) Follistim (follitropin-b, rhFSH produced in CHO cells) Organon Some forms of infertility 1997 (USA) Luveris (lutropin-a ; rhLH produced in CHO cells) Ares-Serono Some forms of infertility 2000 (EU) Ovitrelle also termed Ovidrelle; (rhCG, produced in CHO cells) Serono Used in selected assisted reproductive techniques 2001 (EU), 2000 (USA) Continued APPENDIX 1 501 Product Company Therapeutic indication Date approved Forcaltonin (r salmon calcitonin, produced in E. coli) Unigene Paget’s disease 1999 (EU) Haemopoietic growth factors Epogen (rhEPO, produced in a mammalian cell line) Amgen Treatment of anaemia 1989 (USA) Procrit (rhEPO, produced in a mammalian cell line) Ortho Biotech Treatment of anaemia 1990 (USA) Neorecormon (rhEPO, produced in CHO cells) Boehringer- Mannheim Treatment of anaemia 1997 (EU) Aranesp (darbepoetin-a; long-acting rEPO analogue produced in CHO cells) Amgen Treatment of anaemia 2001 (EU, USA) Nespo (darbepoetin-a; see also Aranesp; long-acting rEPO analogue produced in CHO cells) Dompe Biotec Treatment of anaemia 2001 (EU) Leukine (rGM-CSF, differs from the native human protein by one amino acid, Leu 23. Produced in S. cerevisiae) Immunex Autologous bone marrow transplantation 1991 (USA) Neupogen (Filgrastim, rG-CSF; differs from human protein by containing an additional N-terminal methionine. Produced in E. coli ) Amgen Chemotherapy-induced neutropenia 1991 (USA) Neulasta (PEGfilgrastim, rPEGyl- ated filgrastim — see Neupogen). Also marketed in EU as Neupopeg Amgen Neutropenia 2002 (USA, EU) Recombinant interferons and interleukins Intron A (rIFN-a-2b, produced in E. coli ) Schering Plough Cancer, genital warts, hepatitis 1986 (USA), 2000 (EU) PegIntron A (PEGylated rIFN-a-2b, produced in E. coli ) Schering Plough Chronic hepatitis C 2000 (EU), 2001 (USA) Viraferon (rIFN-a-2b, produced in E. coli ) Schering Plough Chronic hepatitis B and C 2000 (EU) ViraferonPeg (PEGylated rIFN- a-2b, produced in E. coli ) Schering Plough Chronic hepatitis C 2000 (EU) Roferon A (rhIFN-a-2a, produced in E. coli ) Hoffman-La Roche Hairy cell leukaemia 1986 (USA) Actimmune (rhIFN-g-1b, produced in E. coli ) Genentech Chronic granulomatous disease 1990 (USA) Betaferon (rIFN-b-1b, differs from human protein in that Cys 17 is replaced by Ser. Produced in E. coli ) Schering AG Multiple sclerosis 1995 (EU) Betaseron (rIFN-b-1b, differs from human protein in that Cys 17 is replaced by Ser. Produced in E. coli ) Berlex Laboratories and Chiron Relapsing, remitting multiple sclerosis 1993 (USA) 502 BIOPHARMACEUTICALS [...]...APPENDIX 1 Product Avonex (rhIFN-b-1a, produced in CHO cells) Infergen (rIFN-a, synthetic type 1 interferon produced in E coli ) Rebif (rh IFN-b-1a, produced in CHO cells) Rebetron (combination of ribavirin and rhIFN-a-2b produced in E coli ) Alfatronol (rhIFN-a-2b, produced in E coli ) Virtron (rhIFN-a-2b, produced in E coli ) Pegasys (PEGinterferon a-2a, produced in E coli ) Vibragen Omega... Host-cell-derived proteins Host-cell-derived proteins are detected by immunochemical methods, using, for example, polyclonal antisera raised against protein components of the host–vector system used to manufacture the product, unless otherwise prescribed The following types of procedure may be used: liquid-phase displacement assays (e.g radio-immunoassay), liquid-phase direct-binding assays and direct-binding... chromosomal DNA and vector DNA may be separately prepared and used as probes Calibration and standardization Quantitative data are obtained by comparison with responses obtained using standard preparations Chromosomal DNA probes and vector DNA probes are used with chromosomal DNA and vector DNA standards, respectively Standard preparations are calibrated by spectroscopic measurements and stored in a... biotechnology information Biopharmaceuticals: Biochemistry and Biotechnology, Second Edition by Gary Walsh John Wiley & Sons Ltd: ISBN 0 470 84326 8 (ppc), ISBN 0 470 84327 6 (pbk) 510 BIOPHARMACEUTICALS European Federation of Biotechnology (EFB) Site address: http://www.efbweb.org Home page of the EFB, containing information on various facets of biotechnology, including pharmaceutical biotechnology World... possible, and do not cross react with the product Host-cell and vector-derived DNA Residual DNA is detected by hybridization analysis, using suitably sensitive sequence-independent analytical techniques or other suitably sensitive analytical techniques Hybridization analysis DNA in the test sample is denatured to give single-stranded DNA, immobilized on a nitrocellulose or other suitable filter and hybridized... tests, in-process controls and finalproduct tests, for example Amino acid composition Partial amino acid sequence analysis The sequence data permit confirmation of the correct Nterminal processing and detection of the loss of the C-terminal amino acids Peptide mapping Peptide mapping using chemical and/ or enzymatic cleavage of the protein product and analysis by a suitable method such as two-dimensional... position 23 (X1) a2a a2b Lys Arg This monograph applies to interferon-a2a and a2b concentrated solutions The potency of interferon-a2 concentrated solution is not less than 1:4 Â 108 IU per milligram of protein Interferon-a2 concentrated solution contains not less than 2 Â 108 IU of interferon-a2 per millilitre Production Interferon-a2 concentrated solution is produced by a method based on recombinant... solution containing 115 g/l of 522 BIOPHARMACEUTICALS trichloroacetic acid R and 34.5 g/l of sulphosalicylic acid R in water R and agitate the container gently for 60 minutes Transfer the gel to a mixture of 32 volumes of glacial acetic acid R, 100 volumes of ethanol R and 268 volumes of water R, and soak for 5 minutes Immerse the gel for 10 minutes in a staining solution pre-warmed to 608C in which 1.2... less intense bands with molecular masses lower than the principal band No such band is more intense than the principal band in the electropherogram with the reference solution (d) (1%) and not more than three such bands are more intense than the principal band in the electropherogram obtained with reference solution (e) (0.2%) The electropherogram obtained with test solution (a) under non-reducing conditions... under non-reducing conditions may show, in addition to the principal band, less intense bands with molecular masses higher than the principal band No such band is more intense than the principal band in the electropherogram obtained with reference solution (d) (1%) and not more than 3 such bands are more intense than the principal band in the electropherogram obtained with reference solution (e) (0.2%) . (rhIFN-a-2a, produced in E. coli ) Hoffman-La Roche Hairy cell leukaemia 1986 (USA) Actimmune (rhIFN-g-1b, produced in E. coli ) Genentech Chronic granulomatous disease 1990 (USA) Betaferon (rIFN-b-1b,. 1999 (EU) Rebif (rh IFN-b-1a, produced in CHO cells) Ares-Serono Relapsing/remitting multiple sclerosis 1998 (EU), 2002 (USA) Rebetron (combination of ribavirin and rhIFN-a-2b produced in E. coli. USA) Alfatronol (rhIFN-a-2b, produced in E. coli ) Schering Plough Hepatitis B, C, and various cancers 2000 (EU) Virtron (rhIFN-a-2b, produced in E. coli ) Schering Plough Hepatitis B and C 2000 (EU) Pegasys